Bottom Line:
However, increasing MuRF2:PPARγ1 (>5:1) beyond physiological levels drove poly-ubiquitin-mediated degradation of PPARγ1 in vitro, indicating large MuRF2 increases may lead to PPAR degradation if found in other disease states.Mutations in MuRF2 have been described to contribute to the severity of familial hypertrophic cardiomyopathy.These studies also identify MuRF2 as the first ubiquitin ligase to regulate cardiac PPARα and PPARγ1 activities in vivo via post-translational modification without degradation.

Background: In diabetes mellitus the morbidity and mortality of cardiovascular disease is increased and represents an important independent mechanism by which heart disease is exacerbated. The pathogenesis of diabetic cardiomyopathy involves the enhanced activation of PPAR transcription factors, including PPARα, and to a lesser degree PPARβ and PPARγ1. How these transcription factors are regulated in the heart is largely unknown. Recent studies have described post-translational ubiquitination of PPARs as ways in which PPAR activity is inhibited in cancer. However, specific mechanisms in the heart have not previously been described. Recent studies have implicated the muscle-specific ubiquitin ligase muscle ring finger-2 (MuRF2) in inhibiting the nuclear transcription factor SRF. Initial studies of MuRF2-/- hearts revealed enhanced PPAR activity, leading to the hypothesis that MuRF2 regulates PPAR activity by post-translational ubiquitination.

Results: MuRF2 protein levels increase ~20% during the development of diabetic cardiomyopathy induced by high fat diet. Compared to littermate wildtype hearts, MuRF2-/- hearts exhibit an exaggerated diabetic cardiomyopathy, characterized by an early onset systolic dysfunction, larger left ventricular mass, and higher heart weight. MuRF2-/- hearts had significantly increased PPARα- and PPARγ1-regulated gene expression by RT-qPCR, consistent with MuRF2's regulation of these transcription factors in vivo. Mechanistically, MuRF2 mono-ubiquitinated PPARα and PPARγ1 in vitro, consistent with its non-degradatory role in diabetic cardiomyopathy. However, increasing MuRF2:PPARγ1 (>5:1) beyond physiological levels drove poly-ubiquitin-mediated degradation of PPARγ1 in vitro, indicating large MuRF2 increases may lead to PPAR degradation if found in other disease states.

Conclusions: Mutations in MuRF2 have been described to contribute to the severity of familial hypertrophic cardiomyopathy. The present study suggests that the lack of MuRF2, as found in these patients, can result in an exaggerated diabetic cardiomyopathy. These studies also identify MuRF2 as the first ubiquitin ligase to regulate cardiac PPARα and PPARγ1 activities in vivo via post-translational modification without degradation.

Mentions:
Echocardiographic analysis of MuRF2−/− hearts at baseline found no deficits in function or differences in measurements (Fig. 2a; Table 1) as previously described [40, 46]. Significant deficits in heart function were identified in the MuRF2−/− hearts in as little as 6 weeks after the initiation of high fat diet (Fig. 2a, upper left panel). MuRF2−/− hearts were significantly thinner than MuRF2+/+ hearts from 15–26 weeks of high fat diet feeding (Fig. 2a, upper middle panels, Fig. 2b, d). Both MuRF2−/− and wildtype mice experienced an equal progressive dilation over time on a high fat diet, evidenced by increases in LVESD (Fig. 2a, far right panel). MuRF2−/− hearts exhibited significant dysfunction as early as 6 weeks of HFD compared to MuRF2+/+ hearts (Fig. 2b). MuRF2−/− hearts were significantly larger than MuRF2+/+ hearts after 26 weeks high fat diet (Fig. 2b–d). Diabetic cardiomyocyte-related changes in myosin heavy chain gene expression were next investigated to determine differences between groups. Comparable increases in βMHC were seen in MuRF2−/− and wildtype hearts (Fig. 2e), consistent with previous studies identifying these increases [47]. MuRF2−/− cardiac expression of skeletal muscle α-actin and αMHC were increased in chow control hearts compared to wild type mice, and MuRF2−/− skeletal muscle α-actin was significantly increased as compared to wild type mice after 26 weeks high fat diet (Fig. 2e). No difference existed in αMHC after 26 weeks of high fat diet in either MuRF2−/− or MuRF2 +/+ mice (Fig. 2e), although this is reported in other models of diabetic cardiomyopathy [48, 49]. Brain natriuretic protein (BNP) mRNA was decreased in both MuRF2−/− and controls after 26 weeks high fat diet feeding (Fig. 2e). Taken together, these studies identified that MuRF2−/− hearts failed sooner than MuRF2+/+ hearts, resulting in larger hearts, including LV wall thickness and heart weights after 26 weeks high fat diet challenge.Fig. 2

Mentions:
Echocardiographic analysis of MuRF2−/− hearts at baseline found no deficits in function or differences in measurements (Fig. 2a; Table 1) as previously described [40, 46]. Significant deficits in heart function were identified in the MuRF2−/− hearts in as little as 6 weeks after the initiation of high fat diet (Fig. 2a, upper left panel). MuRF2−/− hearts were significantly thinner than MuRF2+/+ hearts from 15–26 weeks of high fat diet feeding (Fig. 2a, upper middle panels, Fig. 2b, d). Both MuRF2−/− and wildtype mice experienced an equal progressive dilation over time on a high fat diet, evidenced by increases in LVESD (Fig. 2a, far right panel). MuRF2−/− hearts exhibited significant dysfunction as early as 6 weeks of HFD compared to MuRF2+/+ hearts (Fig. 2b). MuRF2−/− hearts were significantly larger than MuRF2+/+ hearts after 26 weeks high fat diet (Fig. 2b–d). Diabetic cardiomyocyte-related changes in myosin heavy chain gene expression were next investigated to determine differences between groups. Comparable increases in βMHC were seen in MuRF2−/− and wildtype hearts (Fig. 2e), consistent with previous studies identifying these increases [47]. MuRF2−/− cardiac expression of skeletal muscle α-actin and αMHC were increased in chow control hearts compared to wild type mice, and MuRF2−/− skeletal muscle α-actin was significantly increased as compared to wild type mice after 26 weeks high fat diet (Fig. 2e). No difference existed in αMHC after 26 weeks of high fat diet in either MuRF2−/− or MuRF2 +/+ mice (Fig. 2e), although this is reported in other models of diabetic cardiomyopathy [48, 49]. Brain natriuretic protein (BNP) mRNA was decreased in both MuRF2−/− and controls after 26 weeks high fat diet feeding (Fig. 2e). Taken together, these studies identified that MuRF2−/− hearts failed sooner than MuRF2+/+ hearts, resulting in larger hearts, including LV wall thickness and heart weights after 26 weeks high fat diet challenge.Fig. 2

Bottom Line:
However, increasing MuRF2:PPARγ1 (>5:1) beyond physiological levels drove poly-ubiquitin-mediated degradation of PPARγ1 in vitro, indicating large MuRF2 increases may lead to PPAR degradation if found in other disease states.Mutations in MuRF2 have been described to contribute to the severity of familial hypertrophic cardiomyopathy.These studies also identify MuRF2 as the first ubiquitin ligase to regulate cardiac PPARα and PPARγ1 activities in vivo via post-translational modification without degradation.

Background: In diabetes mellitus the morbidity and mortality of cardiovascular disease is increased and represents an important independent mechanism by which heart disease is exacerbated. The pathogenesis of diabetic cardiomyopathy involves the enhanced activation of PPAR transcription factors, including PPARα, and to a lesser degree PPARβ and PPARγ1. How these transcription factors are regulated in the heart is largely unknown. Recent studies have described post-translational ubiquitination of PPARs as ways in which PPAR activity is inhibited in cancer. However, specific mechanisms in the heart have not previously been described. Recent studies have implicated the muscle-specific ubiquitin ligase muscle ring finger-2 (MuRF2) in inhibiting the nuclear transcription factor SRF. Initial studies of MuRF2-/- hearts revealed enhanced PPAR activity, leading to the hypothesis that MuRF2 regulates PPAR activity by post-translational ubiquitination.

Results: MuRF2 protein levels increase ~20% during the development of diabetic cardiomyopathy induced by high fat diet. Compared to littermate wildtype hearts, MuRF2-/- hearts exhibit an exaggerated diabetic cardiomyopathy, characterized by an early onset systolic dysfunction, larger left ventricular mass, and higher heart weight. MuRF2-/- hearts had significantly increased PPARα- and PPARγ1-regulated gene expression by RT-qPCR, consistent with MuRF2's regulation of these transcription factors in vivo. Mechanistically, MuRF2 mono-ubiquitinated PPARα and PPARγ1 in vitro, consistent with its non-degradatory role in diabetic cardiomyopathy. However, increasing MuRF2:PPARγ1 (>5:1) beyond physiological levels drove poly-ubiquitin-mediated degradation of PPARγ1 in vitro, indicating large MuRF2 increases may lead to PPAR degradation if found in other disease states.

Conclusions: Mutations in MuRF2 have been described to contribute to the severity of familial hypertrophic cardiomyopathy. The present study suggests that the lack of MuRF2, as found in these patients, can result in an exaggerated diabetic cardiomyopathy. These studies also identify MuRF2 as the first ubiquitin ligase to regulate cardiac PPARα and PPARγ1 activities in vivo via post-translational modification without degradation.